Papers by Karla Mayolo-Deloisa
Biotechnology journal, Jan 7, 2018
Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enha... more Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enhance recovery of industrially relevant bioproducts. After ATPS discovery, a variety of works have been published regarding their scaling from 10 to 1000 L. Although ATPS have achieved high recovery and purity yields, there is still a gap between their bench-scale use and potential industrial applications. In this context, this review paper critically analyzes ATPS scale-up strategies to enhance the potential industrial adoption. In particular, large-scale operation considerations, different phase separation procedures, the available optimization techniques (univariate, response surface methodology, and genetic algorithms) to maximize recovery and purity and economic modeling to predict large-scale costs, are discussed. ATPS intensification to increase the amount of sample to process at each system, developing recycling strategies and creating highly efficient predictive models, are still ...
BACKGROUND: Protein refolding represents the limiting step in the recovery of these molecules fro... more BACKGROUND: Protein refolding represents the limiting step in the recovery of these molecules from inclusion bodies and from denaturing operations. This raises the need for establishing different methodologies to efficiently recover and refold proteins. Aqueous two-phase systems (ATPS) besides being a primary recovery operation provide good and affordable physicochemical conditions to refold these molecules.
Protein hydrophobicity can be modified after a PEGylation process. However, hydropho-bic interact... more Protein hydrophobicity can be modified after a PEGylation process. However, hydropho-bic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, b-lactoglobulin, and lysozyme. All proteins were PEGylated in the N-terminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGy-lation reaction mixtures, since the peaks of mono-and di-PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of b-lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC.
The potential recovery of high-value products from brewery yeast waste confers value to this indu... more The potential recovery of high-value products from brewery yeast waste confers value to this industrial residue. Aqueous two-phase systems (ATPS) have demonstrated to be an attractive alternative for the primary recovery of biological products and are therefore suitable for the recovery of invertase from this residue. Sixteen different polyethylene glycol (PEG)–potassium phosphate ATPS were tested to evaluate the effects of PEG molecular weight (MW) and tie-line length (TLL) upon the partition behavior of invertase. Concentrations of crude extract from brewery yeast waste were then varied in the systems that presented the best behaviors to intensify the potential recovery of the enzyme. Results show that the use of a PEG MW 400 g mol −1 system with a TLL of 45.0% (w/w) resulted in an invertase bottom phase recovery with a purification factor of 29.5 and a recovery yield of up to 66.2% after scaling the system to a total weight of 15.0 g. This represents 15.1 mg of invertase per mL of processed bottom phase. With these results, a single-stage ATPS process for the recovery of invertase is proposed.
Journal of Chemical Technology and Biotechnology, Sep 2, 2010
For more than 30 years, PEGylation has been used to improve the physicochemical properties of sev... more For more than 30 years, PEGylation has been used to improve the physicochemical properties of several proteins and therapeutic drugs having a major impact in the biopharmaceutical industry. The purification of PEGylated proteins usually involves two basic challenges: (1) the separation of PEG-proteins from other reaction products; and (2) the sub-fractionation of PEG-proteins on the basis of their degree of PEGylation and positional isomerism. Currently, most PEGylated protein purification processes are based on chromatographic techniques, especially size exclusion chromatography (SEC) and ion exchange chromatography (IEX). Nonetheless, other less frequently used strategies based on non-chromatographic techniques such as ultrafiltration, electrophoresis, capillary electrophoresis, and aqueous two-phase systems have been developed in order to fractionate and analyze PEGylated derivates. This review presents current advances in some of the most widely used non-chromatographic strategies for the fractionation and analysis of PEG-protein conjugates.
Journal of Chemical Technology and Biotechnology, Jun 15, 2015
BACKGROUND: Protein refolding represents the limiting step in the recovery of these molecules fro... more BACKGROUND: Protein refolding represents the limiting step in the recovery of these molecules from inclusion bodies and from denaturing operations. This raises the need for establishing different methodologies to efficiently recover and refold proteins. Aqueous two-phase systems (ATPS) besides being a primary recovery operation provide good and affordable physicochemical conditions to refold these molecules.
RESULTS: 48 different PEG–potassium phosphate ATPS were tested for the partition and refolding of native and denatured invertase. Invertase in both forms has a partition preference for the bottom salt-rich phase where the polymer molecular weight and the tie-line length (TLL) selected for the system play a preponderant role. Refolding percentages of up to 100% can be obtained in the polymer-rich phase and between 50% and 75% in the bottom phase.
CONCLUSIONS: This study reports the characterization of how the different ATPS design parameters such as polymer molecular weight, tie-line length (TLL) and volume ratio (VR ), influence protein partition and refolding using invertase as the protein model. A PEG 3350 g mol−1 , TLL 25.0% w/w and VR = 1.00 system was selected as the best option for denatured invertase recovery and refolding with an enzyme recovery of 60.8% and a refolding percentage of 59.7% in the bottom phase of the system. These findings represent advancements en route to establishing an ATPS operation for the potential in situ recovery and refolding of denatured protein based-products.
Separation and Purification Technology, 2009
PEGylation, used to mitigate some of the problems that affect the effectiveness of therapeutic pr... more PEGylation, used to mitigate some of the problems that affect the effectiveness of therapeutic proteins, often results in a heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. This study presents the use of a mildly amphiphilic support, Tris-capped CH Sepharose 4B as an alternative for separating PEGylated proteins from their unmodified counterparts. The effects of parameters such as pH, salt type and salt concentration upon the chromatographic behavior of native, mono-PEGylated and di-PEGylated ribonuclease A on this media were characterized. The separation of the native protein from the PEGylated species was achieved using a gradient elution between a high ionic strength mobile phase (3 M ammonium sulfate in 25 mM potassium phosphate, pH 7.0 or 2 M potassium phosphate, pH 7.0) and a low ionic strength phase (25 mM potassium phosphate, pH 7.0). The pH of the mobile phases as well as the addition of PEG 600 (as a potential mobile phase modifier) to the low ionic strength phase had no significant influence on chromatographic behavior of the species. This media provides a simple and practical chromatographic method for the separation of unmodified proteins from their corresponding PEG conjugates.
Journal of Chemical Technology & Biotechnology, 2008
The development of recovery processes using aqueous two-phase systems (ATPS) has been limited by ... more The development of recovery processes using aqueous two-phase systems (ATPS) has been limited by the extensive experimental work required to establish the optimal system parameters to ensure selective partitioning of the product of interest. Although using full factorial experiments has been demonstrated to be an effective strategy for the characterization of the partitioning behaviour of biological products in ATPS, this approach is characterized by its costly and time consuming nature. As an alternative, the use of a robotic-aided strategy has been proposed. However, the need for high cost equipment may limit the generic implementation of this strategy. Based on practical experience using ATPS, practical strategies for the predictive design of primary recovery processes using polymer-salt systems were derived. To evaluate the generic application of the proposed strategies, four experimental models (B-phycoerythrin, C-phycocyanin, double layered rotavirus-like particles and lutein) were selected. The application of these strategies resulted in the development of simplified recovery processes for the selected experimental models. The practical review presented is considered a relevant contribution that will provide general rules to facilitate the establishment of ATPS processes, particularly for new researchers in the field.
Process Biochemistry, 2011
The polymer-salt rich environment of aqueous two-phase systems disturbs standard protein quantifi... more The polymer-salt rich environment of aqueous two-phase systems disturbs standard protein quantification methods, like UV measurement or Bradford assay.
Journal of Chemical Technology & Biotechnology, 2011
For more than 30 years, PEGylation has been used to improve the physicochemical properties of sev... more For more than 30 years, PEGylation has been used to improve the physicochemical properties of several proteins and therapeutic drugs having a major impact in the biopharmaceutical industry. The purification of PEGylated proteins usually involves two basic challenges: (1) the separation of PEG-proteins from other reaction products; and (2) the sub-fractionation of PEG-proteins on the basis of their degree of PEGylation and positional isomerism. Currently, most PEGylated protein purification processes are based on chromatographic techniques, especially size exclusion chromatography (SEC) and ion exchange chromatography (IEX). Nonetheless, other less frequently used strategies based on non-chromatographic techniques such as ultrafiltration, electrophoresis, capillary electrophoresis, and aqueous two-phase systems have been developed in order to fractionate and analyze PEGylated derivates. This review presents current advances in some of the most widely used non-chromatographic strategies for the fractionation and analysis of PEG-protein conjugates.
Chemical Engineering & Technology, 2011
Laccase partially purified from residual compost of Agaricus bisporus by an aqueous two-phase sys... more Laccase partially purified from residual compost of Agaricus bisporus by an aqueous two-phase system (Lac ATPS) was used in degrading polycyclic aromatic hydrocarbons: fluorene (Flu), phenanthrene (Phe), anthracene (Ant), benzo[a]pyrene (BaP), and benzo[a]anthracene (BaA). The capacity of the enzyme to oxidize polyaromatic compounds was compared to that of the crude laccase extract (CE). After treatment of 72 h, Lac ATPS and CE were not capable of oxidizing Flu and Phe, while Ant, BaP, and BaA were oxidized, resulting in percentages of oxidation of 11.2 ± 1, 26 ± 2, and 11.7 ± 4 % with CE, respectively. When Lac ATPS was used, the following percentages of oxidation were obtained: 11.4 ± 3 % for Ant, 34 ± 0.1 % for BaP, and 13.6 ± 2 % for BaA. The results reported here demonstrate the potential application of Lac ATPS for the oxidation of polycyclic aromatic hydrocarbons.
Journal of Chromatography A, 2012
The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclu... more The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20 kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1 M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ∼85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.
La PEGilación es la conjugación de una proteína y/o péptido con una o más moléculas de poli(etile... more La PEGilación es la conjugación de una proteína y/o péptido con una o más moléculas de poli(etilen glicol). El poli(etilen glicol) es un polímero no tóxico, no inmunogénico y está aprobado por la FDA (Food and Drug Administration, USA). En losúltimos años, la PEGilación ha sido utilizada para mejorar las propiedades fisicoquímicas de proteínas y drogas terapéuticas, por lo que esta tecnología ha impactado fuertemente a la industria bio-farmacéutica. La PEGilación permite prolongar el tiempo de residencia en el cuerpo, mejorar la estabilidad, aumentar la solubilidad, disminuir la proteólisis y excreción renal. Desde el surgimiento de esta tecnología, diferentes proteínas han sido PEGiladas para el tratamiento de enfermedades como: hepatitis C, leucemia, artritis reumatoide, etc. Este artículo de revisión presenta una descripción del desarrollo de la PEGilación en losúltimos años, así como de los procedimientos usados para la producción de bio-conjugados. Además, se revisan las estrategias de purificación utilizadas para la recuperación de proteínas PEGiladas, siendo este uno de los grandes retos en el proceso debido a que la reacción de PEGilación puede generar bio-conjugados con diferentes grados de PEGilación. Porúltimo, se presentan las aplicaciones de dichos bio-conjugados y los retos futuros que se identifican para su aplicación genérica.
Journal of Molecular Recognition, 2015
Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can ... more Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme. Copyright © 2015 John Wiley & Sons, Ltd.
Journal of Chromatography A, 2014
The covalent attachment of polyethylene glycol (PEG) molecules to pharmaceutical proteins, "PEGyl... more The covalent attachment of polyethylene glycol (PEG) molecules to pharmaceutical proteins, "PEGylation", often results in a population of conjugate species that includes differing numbers and locations of attached PEG chains. As some portion of this population may be biologically inactive, a challenging separation problem arises. An interesting alternative to the size-based resolution of these conjugates involves the use of reversed phase chromatography (RPC), treating the PEG moieties as hydrophobic purification tags. The use of RPC raises concerns about protein denaturation in the mobile and on the stationary phase. Here, the potential dual role of conjugated PEG chains as both group-specific separation tags and as steric or structural stabilizers in RPC was explored. In this work, RPC with C18-based media was used to resolve PEGylation number variants of ribonuclease A (RNase A) and apo-␣-lactalbumin (apo-␣Lac) in a neutral pH mobile phase. While the attachment of 20 kDa PEG molecules did not modify the structures of RNase A and apo-␣Lac, as confirmed by structural analysis using circular dichroism, exposure to the mobile phase modifier, acetonitrile, and to the C18 media during separation resulted in perturbations to both the secondary and tertiary structures of all species studied. RNase A experienced small perturbations that were mediated to some extent by PEGylation; these results were consistent with activity assays which showed that PEGylated RNase A species retained native-like activity after RPC separation. Apo-␣Lac, a more hydrophobic and less stable protein than RNase A, experienced extensive structural perturbations regardless of PEGylation state. The temperature of the mobile phase was found to strongly influence chromatographic separation of PEG-conjugates with conjugate species becoming more strongly retained with increasing temperature. This work shows that it is feasible to employ RPC with neutral pH mobile phases to resolve PEG conjugate number heterogeneity.
Modelado de la biodegradación en biorreactores de lodos de hidrocarburos totales del petróleo int... more Modelado de la biodegradación en biorreactores de lodos de hidrocarburos totales del petróleo intemperizados en suelos y sedimentos (Biodegradation modeling of sludge bioreactors of total petroleum hydrocarbons weathering in soil and sediments)
La PEGilación es la conjugación de una proteína y/o péptido con una o más moléculas de poli(etile... more La PEGilación es la conjugación de una proteína y/o péptido con una o más moléculas de poli(etilen glicol). El poli(etilen glicol) es un polímero no tóxico, no inmunogénico y está aprobado por la FDA (Food and Drug Administration, USA). En losúltimos años, la PEGilación ha sido utilizada para mejorar las propiedades fisicoquímicas de proteínas y drogas terapéuticas, por lo que esta tecnología ha impactado fuertemente a la industria bio-farmacéutica. La PEGilación permite prolongar el tiempo de residencia en el cuerpo, mejorar la estabilidad, aumentar la solubilidad, disminuir la proteólisis y excreción renal. Desde el surgimiento de esta tecnología, diferentes proteínas han sido PEGiladas para el tratamiento de enfermedades como: hepatitis C, leucemia, artritis reumatoide, etc. Este artículo de revisión presenta una descripción del desarrollo de la PEGilación en losúltimos años, así como de los procedimientos usados para la producción de bio-conjugados. Además, se revisan las estrategias de purificación utilizadas para la recuperación de proteínas PEGiladas, siendo este uno de los grandes retos en el proceso debido a que la reacción de PEGilación puede generar bio-conjugados con diferentes grados de PEGilación. Porúltimo, se presentan las aplicaciones de dichos bio-conjugados y los retos futuros que se identifican para su aplicación genérica.
PEGylation, used to mitigate some of the problems that affect the effectiveness of therapeutic pr... more PEGylation, used to mitigate some of the problems that affect the effectiveness of therapeutic proteins, often results in a heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. This study presents the use of a mildly amphiphilic support, Tris-capped CH Sepharose 4B as an alternative for separating PEGylated proteins from their unmodified counterparts. The effects of parameters such as pH, salt type and salt concentration upon the chromatographic behavior of native, mono-PEGylated and di-PEGylated ribonuclease A on this media were characterized. The separation of the native protein from the PEGylated species was achieved using a gradient elution between a high ionic strength mobile phase (3 M ammonium sulfate in 25 mM potassium phosphate, pH 7.0 or 2 M potassium phosphate, pH 7.0) and a low ionic strength phase (25 mM potassium phosphate, pH 7.0). The pH of the mobile phases as well as the addition of PEG 600 (as a potential mobile phase modifier) to the low ionic strength phase had no significant influence on chromatographic behavior of the species. This media provides a simple and practical chromatographic method for the separation of unmodified proteins from their corresponding PEG conjugates.
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Papers by Karla Mayolo-Deloisa
RESULTS: 48 different PEG–potassium phosphate ATPS were tested for the partition and refolding of native and denatured invertase. Invertase in both forms has a partition preference for the bottom salt-rich phase where the polymer molecular weight and the tie-line length (TLL) selected for the system play a preponderant role. Refolding percentages of up to 100% can be obtained in the polymer-rich phase and between 50% and 75% in the bottom phase.
CONCLUSIONS: This study reports the characterization of how the different ATPS design parameters such as polymer molecular weight, tie-line length (TLL) and volume ratio (VR ), influence protein partition and refolding using invertase as the protein model. A PEG 3350 g mol−1 , TLL 25.0% w/w and VR = 1.00 system was selected as the best option for denatured invertase recovery and refolding with an enzyme recovery of 60.8% and a refolding percentage of 59.7% in the bottom phase of the system. These findings represent advancements en route to establishing an ATPS operation for the potential in situ recovery and refolding of denatured protein based-products.
RESULTS: 48 different PEG–potassium phosphate ATPS were tested for the partition and refolding of native and denatured invertase. Invertase in both forms has a partition preference for the bottom salt-rich phase where the polymer molecular weight and the tie-line length (TLL) selected for the system play a preponderant role. Refolding percentages of up to 100% can be obtained in the polymer-rich phase and between 50% and 75% in the bottom phase.
CONCLUSIONS: This study reports the characterization of how the different ATPS design parameters such as polymer molecular weight, tie-line length (TLL) and volume ratio (VR ), influence protein partition and refolding using invertase as the protein model. A PEG 3350 g mol−1 , TLL 25.0% w/w and VR = 1.00 system was selected as the best option for denatured invertase recovery and refolding with an enzyme recovery of 60.8% and a refolding percentage of 59.7% in the bottom phase of the system. These findings represent advancements en route to establishing an ATPS operation for the potential in situ recovery and refolding of denatured protein based-products.